The long-term objective of this project is to understand the molecular mechanism of homologous recombination initiation during meiosis. Recombination is essential for meiosis in most sexually reproducing organisms, including humans, because it generates genetic diversity and ensures the accurate segregation of homologous chromosomes. In S. cerevisiae, meiotic recombination initiates via the formation of DNA double-strand breaks (DSBs) through a transesterification reaction catalyzed by Spo11, a member of a widely conserved family that includes meiotic recombination proteins from higher eukaryotes and type II topoisomerases of archaebacteria. This study will use Spo11 as the focal point to investigate the molecular mechanism of DSB formation. The Specific Aims are: l. To identify proteins that interact with Spo11. At least nine gene products in addition to Spo11 are required for DSB formation, suggesting that physical interactions between Spo11 and other proteins are important for Spo11 function. These interactions will be identified using several complementary approaches: a) Spo11-containing complexes will be affinity-purified from extracts of meiotic cells, and the protein components will be identified by mass spectrometric tryptic peptide fingerprinting. b) Proteins required for DSB formation will be coexpressed with Spo11 in vitro and assayed for complex formation. And c) yeast two-hybrid screens with Spoil as bait will be carried out. 2. To characterize the activities of Spoil and the proteins that interact with Spo11. Spo11 will be over-expressed and purified, and will be examined for DNA binding, DNA cleavage, and topoisomerase activities. Proteins found to interact with Spo11 will also be overexpressed and purified. Their activities and their effects on the activities of Spo11 will be characterized. 3. To develop an in vitro assay system for DSB formation. Recent evidence indicates that chromatin and higher order structures of meiotic chromosomes influence the locations and frequencies of DSBs. To begin to address the molecular basis of these effects, a biochemically accessible system that uses whole meiotic chromosomes as substrates for DSB formation will be developed. Conditions have been identified in which nuclei isolated from wild-type meiotic cells are competent to form new DSBs in vitro. This system will be optimized and characterized, then the biochemical components will be dissected by fractionation of wild- type nuclei, and by adaptation for in vitro complemenation of nuclei isolated from DSB-defective mutant cells.